Enzymes are used on an industrial scale as catalysts for processing various crude materials. Often these processes are cost-effective only when the enzymes can be re-used many times. For recirculation the enzymes need to be separated from the process liquid. This is possible when the enzymes are attached to a carrier which can be filtrated or centrifuged.
An important group of industrial enzymes have an amphiphilic nature. Lipases and phospholipases belong to this group of enzymes, which are characterized by the presence of a hydrophilic part as well as a hydrophobic part in the molecule. Such enzymes, when dispersed in an emulsion, will migrate and accumulate in the interface of the aqueous and the oil phase. The hydrophobic part of the enzyme points into the hydrophobic phase and the hydrophilic part points into the aqueous phase.
The invention will be described with lipase as an example of an amphiphilic enzyme. Another industrially important amphiphilic enzyme is phospholipase of which various types are known and which is used for the hydrolysis of phospholipids to lysophospholipids. An industrial application is the enzymatic degumming of triglyceride oils, which is described in e.g. EP 513 709.
Lipases are employed for their ability to modify the structure and composition of triglyceride oils and fats. They catalyze different types of triglyceride conversions, such as hydrolysis, esterification and transesterification. These are equilibrium reactions which in one direction result into hydrolysis of triglycerides into free fatty acids and glycerol, mono- or diglycerides, and in the other direction result into re-esterification of glycerol, monoglycerides and diglycerides into triglycerides. For the re-esterification process removal of the water in the reaction medium is necessary to shift the equilibrium in the direction of the triglyceride synthesis.
The use of lipase in substantially water-free process media is associated with a major problem which is the solubilization of lipase in oil. For that purpose preferably a lipase is used which is active in a water-free environment and which is immobilized.
Presently the main process for lipase manufacture is a microbiological fermentation of suitable microorganisms which produce the enzyme under proper conditions. To a purified lipase solution a carrier is added and the enzyme is allowed to get attached to the carrier surface. Such immobilization method is exemplified for an interesterification process in e.g. GB 2 159 527. The attachment of the enzyme to the carrier enables easy separation of the irreversibly immobilized enzyme from the process medium for subsequent use.
Generally, the used carrier materials are porous, particulate, but always water insoluble materials which provide large surface areas per unit volume are. The preparation of immobilized enzymes is described in e.g. EP 0 140 542, EP 0 382 767, WO 95/22606, EP 0 444 092and WO 89/01032.
Since the present immobilisation techniques require pure enzyme, the crude fermentation broth has to be subjected to an extensive and costly purification process which is a disadvantage inherent to the present production of immobilised enzyme systems.
Moreover, to the extent that the active parts of the immobilized enzyme are facing the carrier material rather than being exposed to the substrate, the activity will be correspondingly low. In the usual porous carrier materials mass transfer effects further limit the lipase activity.
The present invention aims to provide a process for immobilizing an enzyme in which a non-purified enzyme preparation may be utilized. Furthermore the present invention aims to provide a highly active, immobilized enzyme preparation which can be regenerated and reactivated.